transport Virtually all eukaryotic cells transport organelles and a subset of mRNA molecules to specific destinations within the cytoplasm. Such transport generates asymmetric distributions of macromolecules, which is critical for establishing the body plan during early development and for establishing cell polarity and specialized intracellular domains. Hence, deciphering the mechanism of intracellular transport is fundamental for understanding many basic biological functions. Medical implications of this work include understanding and treating neurodegenerative diseases (some of which may be due to impaired transport), understanding the basis of development defects, and designing strategies for delivering drugs or protein therapeutics within cells. Most intracellular transport involves the movement of motor proteins along a cytoskeletal track. In the last few years, many types of motor proteins have been discovered (e.g. members of the kinesin or myosin superfamily) and linked to particular intracellular transport events. However, the macromolecules that mediate the interactions between motors and their cargo are less well understood. We seek to study three types of macromolecular complexes that are involved in transport by molecular motors: 1) RNP complexes that is transported by myosin motor proteins in S. cerevisiae, 2) kinesin motor protein that interacts with lipids and proteins on membrane vesicles, and 3) a microtubule end binding complex that helps to bring microtubules into contact with motor proteins anchored to the cell cortex. We will investigate these complexes using biochemical and structural approaches. Overall, the experiments in this proposal are designed to identify all proteins at the motor- cargo interface, dissect their functions through in vitro and vivo experiments, and determine the structures of the complex and/or key components of the complex. These studies will provide a mechanistic understanding of how cargoes bind to specific motors and how cells asymmetrically position components within their cytoplasm.